Differential, dominant activation and inhibition of Notch signalling and APP cleavage by truncations of PSEN1 in human disease.

PRESENILIN1 (PSEN1) is the major locus for mutations causing familial Alzheimer's disease (FAD) and is also mutated in Pick disease of brain, familial acne inversa and dilated cardiomyopathy. It is a critical facilitator of Notch signalling and many other signalling pathways and protein cleavage events including production of the Amyloidß (Aß) peptide from the AMYLOID BETA A4 PRECURSOR PROTEIN (APP). We previously reported that interference with splicing of transcripts of the zebrafish orthologue of PSEN1 creates dominant negative effects on Notch signalling. Here, we extend this work to show that various truncations of human PSEN1 (or zebrafish Psen1) protein have starkly differential effects on Notch signalling and cleavage of zebrafish Appa (a paralogue of human APP). Different truncations can suppress or stimulate Notch signalling but not Appa cleavage and vice versa. The G183V mutation possibly causing Pick disease causes production of aberrant transcripts truncating the open reading frame after exon 5 sequence. We show that the truncated protein potentially translated from these transcripts avidly incorporates into very stable Psen1-dependent higher molecular weight complexes and suppresses cleavage of Appa but not Notch signalling. In contrast, the truncated protein potentially produced by the P242LfsX11 acne inversa mutation has no effect on Appa cleavage but, unexpectedly, enhances Notch signalling. Our results suggest novel hypotheses for the pathological mechanisms underlying these diseases and illustrate the importance of investigating the function of dominant mutations at physiologically relevant expression levels and in the normally heterozygous state in which they cause human disease rather than in isolation from healthy alleles.

A cell autonomous role for the Notch ligand Delta-like 3 in αß T-cell development.

Notch signalling is critical to help direct T-cell lineage commitment in early T-cell progenitors and in the development of αß T-cells. Epithelial and stromal cell populations in the thymus express the Notch DSL (Delta, Serrate and Lag2)ligands Delta-like 1 (Dll1), Delta-like 4 (Dll4), Jagged 1 and Jagged 2, and induce Notch signalling in thymocytes that express the Notch receptor. At present there is nothing known about the role of the Delta-like 3 (Dll3) ligand in the immune system. Here we describe a novel cell autonomous role for Dll3 in αß T-cell development. We show that Dll3 cannot activate Notch when expressed in trans but like other Notch ligands it can inhibit Notch signalling when expressed in cis with the receptor. The loss of Dll3 leads to an increase in Hes5 expression in double positive thymocytes and their increased production of mature CD4(+) and CD8(+) T cells. Studies using competitive irradiation chimeras proved that Dll3 acts in a cell autonomous manner to regulate positive selection but not negative selection of autoreactive T cells. Our results indicate that Dll3 has a unique function during T-cell development that is distinct from the role played by the other DSL ligands of Notch and is in keeping with other recent studies indicating that Dll1 and Dll3 ligands have non-overlapping roles during embryonic development.

Grb7 SH2 domain structure and interactions with a cyclic peptide inhibitor of cancer cell migration and proliferation.

BACKGROUND: Human growth factor receptor bound protein 7 (Grb7) is an adapter protein that mediates the coupling of tyrosine kinases with their downstream signaling pathways. Grb7 is frequently overexpressed in invasive and metastatic human cancers and is implicated in cancer progression via its interaction with the ErbB2 receptor and focal adhesion kinase (FAK) that play critical roles in cell proliferation and migration. It is thus a prime target for the development of novel anti-cancer therapies. Recently, an inhibitory peptide (G7-18NATE) has been developed which binds specifically to the Grb7 SH2 domain and is able to attenuate cancer cell proliferation and migration in various cancer cell lines. RESULTS: As a first step towards understanding how Grb7 may be inhibited by G7-18NATE, we solved the crystal structure of the Grb7 SH2 domain to 2.1 A resolution. We describe the details of the peptide binding site underlying target specificity, as well as the dimer interface of Grb 7 SH2. Dimer formation of Grb7 was determined to be in the muM range using analytical ultracentrifugation for both full-length Grb7 and the SH2 domain alone, suggesting the SH2 domain forms the basis of a physiological dimer. ITC measurements of the interaction of the G7-18NATE peptide with the Grb7 SH2 domain revealed that it binds with a binding affinity of Kd = approximately 35.7 microM and NMR spectroscopy titration experiments revealed that peptide binding causes perturbations to both the ligand binding surface of the Grb7 SH2 domain as well as to the dimer interface, suggesting that dimerisation of Grb7 is impacted on by peptide binding. CONCLUSION: Together the data allow us to propose a model of the Grb7 SH2 domain/G7-18NATE interaction and to rationalize the basis for the observed binding specificity and affinity. We propose that the current study will assist with the development of second generation Grb7 SH2 domain inhibitors, potentially leading to novel inhibitors of cancer cell migration and invasion.

N and C-terminal sub-regions in the c-Myc transactivation region and their joint role in creating versatility in folding and binding.

The proto-oncogene c-myc governs the expression of a number of genes targeting cell growth and apoptosis, and its expression levels are distorted in many cancer forms. The current investigation presents an analysis by proteolysis, circular dichroism, fluorescence and Biacore of the folding and ligand-binding properties of the N-terminal transactivation domain (TAD) in the c-Myc protein. A c-Myc sub-region comprising residues 1-167 (Myc1-167) has been investigated that includes the unstructured c-Myc transactivation domain (TAD, residues 1-143) together with a C-terminal segment, which appears to promote increased folding. Myc1-167 is partly helical, binds both to the target proteins Myc modulator-1 (MM-1) and TATA box-binding protein (TBP), and displays the characteristics of a molten globule. Limited proteolysis divides Myc1-167 in two halves, by cleaving in a predicted linker region between two hotspot mutation regions: Myc box I (MBI) and Myc box II (MBII). The N-terminal half (Myc1-88) is unfolded and does not alone bind to target proteins, whereas the C-terminal half (Myc92-167) has a partly helical fold and specifically binds both MM-1 and TBP. Although this might suggest a bipartite organization in the c-Myc TAD, none of the N and C-terminal fragments bind target protein with as high affinity as the entire Myc1-167, or display molten globule properties. Furthermore, merely linking the MBI with the C-terminal region, in Myc38-167, is not sufficient to achieve binding and folding properties as in Myc1-167. Thus, the entire N and C-terminal regions of c-Myc TAD act in concert to achieve high specificity and affinity to two structurally and functionally orthogonal target proteins, TBP and MM-1, possibly through a mechanism involving molten globule formation. This hints towards understanding how binding of a range of targets can be accomplished to a single transactivation domain.

CSL: a notch above the rest.

CSL (CBF1, Suppressor of Hairless, Lag-1) is a transcription factor that is responsible for activating the genes downstream of the Notch signalling pathway, a pathway that is essential for the development of the nervous system and the differentiation of the haematopoietic system among others. In the absence of Notch signalling, CSL represses transcription of Notch target genes, and following activation by Notch, CSL is converted into a transcriptional activator and activates transcription of the same genes. These two opposing functions of CSL are mediated through interactions with distinct protein complexes. The Notch signalling pathway and its crucial cofactor CSL can maintain cells in an undifferentiated state, and have therefore been associated with a growing list of cancers. In addition, CSL has been co-opted by Epstein-Barr virus to mediate viral and host gene transcription following infection.

The new MATH: homology suggests shared binding surfaces in meprin tetramers and TRAF trimers.

Although apparently functionally unrelated, intracellular TRAFs and extracellular meprins share a region with conserved meprin and traf homology, MATH(1). Both TRAFs and meprins require subunit assembly for function. By structural analysis of the sequences, we provide an explanation of how meprins, which form tetramers, and TRAF molecules, which form trimers, can share homology. Our analysis suggests it is highly likely that the same oligomerization surface is used. The analysis has implications for the widely distributed group of proteins containing MATH domains.

NMR analysis of in vitro-synthesized proteins without purification: a high-throughput approach.

A cell-free protein expression system was established that provides protein samples of adequate concentration and purity for direct NMR analysis. The Escherichia coli peptidyl-prolyl cis-trans isomerase PpiB was expressed in this system with dual amino acid-selective isotope labeling to identify the NMR signal from the active site-residue Arg87. Addition of the substrate succinyl-Ala-Ala-Pro-Phe-p-nitroanilide selectively shifted its (15)N-HSQC cross peak, confirming binding to the active site. As cell-free protein expression provides high yields of protein per unit mass of labeled amino acid and sample handling is minimal, this strategy presents an exceptionally inexpensive and rapid approach to protein analysis.

The solution structure and intramolecular associations of the Tec kinase SRC homology 3 domain.

Tec is the prototypic member of a family of intracellular tyrosine kinases that includes Txk, Bmx, Itk, and Btk. Tec family kinases share similarities in domain structure with Src family kinases, but one of the features that differentiates them is a proline-rich region (PRR) preceding their Src homology (SH) 3 domain. Evidence that the PRR of Itk can bind in an intramolecular fashion to its SH3 domain and the lack of a regulatory tyrosine in the C terminus indicates that Tec kinases must be regulated by a different set of intramolecular interactions to the Src kinases. We have determined the solution structure of the Tec SH3 domain and have investigated interactions with its PRR, which contains two SH3-binding sites. We demonstrate that in vitro, the Tec PRR can bind in an intramolecular fashion to the SH3. However, the affinity is lower than that for dimerization via reciprocal PRR-SH3 association. Using site-directed mutagenesis we show that both sites can bind the Tec SH3 domain; site 1 (155KTLPPAP161) binds intramolecularly, while site 2 (165KRRPPPPIPP174) cannot and binds in an intermolecular fashion. These distinct roles for the SH3 binding sites in Tec family kinases could be important for protein targeting and enzyme activation.

Biophysical properties of regions flanking the bHLH-Zip motif in the p22 Max protein.

The Max protein is the central dimerization partner in the Myc-Max-Mad network of transcriptional regulators, and a founding structural member of the family of basic-helix-loop-helix (bHLH)-leucine zipper (Zip) proteins. Biologically important regions flanking its bHLH-Zip motif have been disordered or absent in crystal structures. The present study shows that these regions are resistant to proteolysis in both the presence and absence of DNA, and that Max dimers containing both flanking regions have significantly higher helix content as measured by circular dichroism than that predicted from the crystal structures. Nuclear magnetic resonance measurements in the absence of DNA also support the inferred structural order. Deletion of both flanking regions is required to achieve maximal DNA affinity as measured by EMSA. Thus, the previously observed functionalities of these Max regions in DNA binding, phosphorylation, and apoptosis are suggested to be linked to structural properties.

Partial purification and characterization of gamma-secretase from post-mortem human brain.

One characteristic feature of Alzheimer's disease is the deposition of amyloid beta-peptide (Abeta) as amyloid plaques within specific regions of the human brain. Abeta is derived from the amyloid beta-peptide precursor protein (beta-APP) by the intramembranous cleavage activity of gamma-secretase. Studies in cells have revealed that gamma-secretase is a large multimeric membrane-bound protein complex that is functionally dependent on several proteins, including presenilin, nicastrin, Aph-1, and Pen-2. However, the precise biochemical and molecular nature of gamma-secretase is as yet to be fully elucidated, and no investigations have analyzed gamma-secretase in human brain. To address this we have developed a novel in vitro gamma-secretase activity assay using detergent-solubilized cell membranes and a beta-APP-derived fluorescent probe. We report that human brain-derived gamma-secretase activity co-purifies with a high molecular weight protein complex comprising presenilin, nicastrin, Aph-1, and Pen-2. The inhibitor profile and solubility characteristics of brain-derived gamma-secretase are similar to those described in cells, and proteolysis occurs at the Abeta40- and Abeta42-generating cleavage sites. The ability to isolate gamma-secretase from post-mortem human brain may facilitate the identification of brain-specific modulators of beta-APP processing and provide new insights into the biology of this important factor in the pathogenesis of Alzheimer's disease.

Pen-2 is sequestered in the endoplasmic reticulum and subjected to ubiquitylation and proteasome-mediated degradation in the absence of presenilin.

The gamma-secretase complex catalyzes intramembrane proteolysis of a number of transmembrane proteins, including amyloid precursor protein, Notch, ErbB4, and E-cadherin. gamma-Secretase is known to contain four major protein constituents: presenilin (PS), nicastrin, Aph-1, and Pen-2, all of which are integral membrane proteins. There is increasing evidence that the formation of the complex and the stability of the individual components are tightly controlled in the cell, assuring correct composition of functional complexes. In this report, we investigate the topology, localization, and mechanism for destabilization of Pen-2 in relation to PS function. We show that PS1 regulates the subcellular localization of Pen-2: in the absence of PS, Pen-2 is sequestered in the endoplasmic reticulum (ER) and not transported to post-ER compartments, where the mature gamma-secretase complexes reside. PS deficiency also leads to destabilization of Pen-2, which is alleviated by proteasome inhibitors. In keeping with this, we show that Pen-2, which adopts a hairpin structure with the N and C termini facing the luminal space, is ubiquitylated prior to degradation and presumably retrotranslocated from the ER to the cytoplasm. Collectively, our data suggest that failure to become incorporated into the gamma-secretase complex leads to degradation of Pen-2 through the ER-associated degradation-proteasome pathway.

Nicastrin, presenilin, APH-1, and PEN-2 form active gamma-secretase complexes in mitochondria.

Mitochondria are central in the regulation of cell death. Apart from providing the cell with ATP, mitochondria also harbor several death factors that are released upon apoptotic stimuli. Alterations in mitochondrial functions, increased oxidative stress, and neurons dying by apoptosis have been detected in Alzheimer's disease patients. These findings suggest that mitochondria may trigger the abnormal onset of neuronal cell death in Alzheimer's disease. We previously reported that presenilin 1 (PS1), which is often mutated in familial forms of Alzheimer's disease, is located in mitochondria and hypothesized that presenilin mutations may sensitize cells to apoptotic stimuli at the mitochondrial level. Presenilin forms an active gamma-secretase complex together with Nicastrin (NCT), APH-1, and PEN-2, which among other substrates cleaves the beta-amyloid precursor protein (beta-APP) generating the amyloid beta-peptide and the beta-APP intracellular domain. Here we have identified dual targeting sequences (for endoplasmic reticulum and mitochondria) in NCT and showed expression of NCT in mitochondria by immunoelectron microscopy. We also showed that NCT together with APH-1, PEN-2, and PS1 form a high molecular weight complex located in mitochondria. gamma-secretase activity in isolated mitochondria was demonstrated using C83 (alpha-secretase-cleaved C-terminal 83-residue beta-APP fragment from BD8 cells lacking presenilin and thus gamma-secretase activity) or recombinant C100-Flag (C-terminal 100-residue beta-APP fragment) as substrates. Both systems generated an APP intracellular domain, and the activity was inhibited by the gamma-secretase inhibitors l-685,458 or Compound E. This novel localization of NCT, PS1, APH-1, and PEN-2 expands the role and importance of gamma-secretase activity to mitochondria.